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水稻条纹叶枯病毒(rice stripe virus,RSV)是柔丝病毒组的典型代表,其基因组由4种ss-RNA及相应低含量的4种dsRNA组成,其中,RNA1为负链性质,编码RNA多聚酶;RNA2～4均为双义编码性质(ambisense-coding strategy)。为研究该病毒RNA非编码区等调控元件在病毒基因组双义表达过程中的功能,采用反转录-聚合酶链式反应方法(RT-PCR),扩增出覆盖RSV RNA2 5′末端非编码区、vRNA2OrFT区、基因间非编码区及vcRNA2 ORF部分区域的REPI片段(1 159bp)。将此扩增产物克隆于载体pGEM-7Zf(+)的Sma Ⅰ位点上并进行了核苷酸序列测定。结果表明,中国分离物与日本分离物的REPI片段具极高的序列同源性。将REPI片段克隆到原核高效表达载体pJW2的P_RP_L启动子下游,经温度诱导,获得了高效
Rice stripe virus (RSV) is a typical representative of the genus Rousy virus group. Its genome consists of four kinds of ss-RNAs and four low-content dsRNAs, of which RNA1 is negative-chain, and encodes RNA polymerase ; RNA2 ~ 4 are ambisense-coding strategies. In order to study the function of regulatory elements such as the non-coding region of the viral RNA in the double-sense expression of the viral genome, a reverse transcription-polymerase chain reaction (RT-PCR) Region, vRNA2OrFT region, intergenic non-coding region and part of the vcRNA2 ORF region of the REPI fragment (1 159bp). The amplified product was cloned into SmaI site of vector pGEM-7Zf (+) and the nucleotide sequence was determined. The results showed that the REPI fragments of Chinese isolates and Japanese isolates had very high sequence homology. The REPI fragment was cloned downstream of the P_RP_L promoter of prokaryotic expression vector pJW2 and induced by temperature,